1. Field of the Invention
This invention relates generally to a method for collection of aerosolized proteins for biochemical analysis. In particular, the invention relates to a method for collection of aerosolized therapeutic proteins which are administered to a patient by aerosol. The collected therapeutic protein can be subjected to biochemical analysis to determine the effect that aerosolization has on the activity and integrity of the protein.
2. Description of Related Art
There has been an increase in the number of therapeutic proteins which have shown promise for administration by aerosol to the lungs of the patient for either local action or systemic absorption. For a review of these, see Niven R. W., Pharm Tech. 17:72-82 (1993). Recombinant human deoxyribonuclease I (rhDNase) may be administered as an aerosol to the lungs of patients suffering from cystic fibrosis. rhDNase is able to cleave DNA present in the thick, viscous secretions in the airways of the lungs of the patient thereby reducing sputum viscosity (see, e.g., Shak, S., et al., Proc. Nat. Acad. Sci. 87:9188-92 [1990]; Aitken, M. A., et al., Jama 267:1947-1951 [1992] and Hubbard, R. C., et al., New. Eng. J. Med. 326:812-815 [1992]). Vogelmeier, et al., J. Appl. Physiol. 69(5):1843-1848 (1990) and Hubbard, R. C., et al., Pro. Nat. Acad. Sci. 86: 680-684 (1989) discuss the neutrophil inhibitors; secretory leukoprotease inhibitor (SLPI) and alpha-1-antitrypsin, and their therapeutic potential when administered by aerosol via the respiratory route. U.S. Pat. No. 5,230,884 discloses an aerosol formulation for delivery of insulin to a patient's lungs.
It is clearly desirable to assess the effect aerosolization has on protein activity and integrity. This is especially important where the protein is to be used as a therapeutic. As discussed in Byron, P., Advanced Drug Delivery Reviews, 5: 107-132 (1990), aerosolization of aqueous solutions by nebulizer generates high shear forces which can denature proteins. Also, because of the surface-active nature of proteins, surface fouling and foaming of the protein can occur during nebulization of a protein solution. Oeswein et al. discuss several concerns relating to the use of aerosol generators [Proceedings of the Second Respiratory Drug Delivery Symposium; Dalby, R. N.; Evans, R., Eds.; Continuing Pharmacy Education, University of Kentucky: KY, pp. 14-49 (1991)]. These concerns relate to potential drug denaturation or inactivation, dose delivery efficiency and accuracy, drug stability, particle size, safety, toxicity and pharmacokinetic efficacy. A further concern with respect to non-aqueous aerosols relates to dispersibility of the protein. Oeswein et al. found that, upon aerosolization, human growth hormone (hGH) tends to form metastable, partially-unfolded intermediates which have a high tendency to aggregate. This is considered to be quite problematic, especially if aggregation results in a decrease in bioactivity, or otherwise affects immunogenicity or safety of the therapeutic protein.
There is little information in the literature concerning the effect aerosolization has on protein activity and integrity. Mercer et al. discuss capturing aerosolized small molecular weight drugs, such as sodium chloride and cesium chloride, and then drying these molecules for subsequent drug characterization (Mercer, T. T. et al., Am. Ind. Hyg. Assoc. J. 29: 66-78 [1968]). This procedure is not suitable for proteins which may adhere to the membrane filter collection surface and thereby denature.
Oeswein et al., supra, generated an aerosol of hGH solution using either a Turret.RTM. or an Acorn-II.RTM. nebulizer (both by Marquest) and the aerosolized protein so generated was collected by impaction on a test tube. From studies of the UV spectrum of the collected hGH it was shown that extensive non-covalent aggregation of protein had occurred (evidenced by light scattering). While Oswein and his colleagues did not discuss the percentage of hGH recovered, in the experiments disclosed herein, it was found that impaction on a test tube results in quite variable and low recovery efficiencies of the protein.
Hubbard et al. (1989), supra, studied the effect that aerosolization had on recombinant human .alpha..sub.1 -antitrypsin (rAAT) aerosol in vitro. An aerosol of a solution of rAAT in physiologic saline solution was generated at a rate of 10 liters/min using an Ultravent nebulizer (by Mallinckrodt). The aerosol was collected by bubbling the nebulizer output through phosphate-buffered saline (at pH 7.4) and the resultant fluid was concentrated by pressure filtration using a UM10 membrane (by Amicon). The structural and functional effects of nebulization on the protein were measured. To measure any effect nebulization had on the integrity of the rAAT, SDS-PAGE gels of rAAT before, and after, aerosolization were compared. The association rate constant (K.sub.a) of rAAT for human neutrophil elastase was also measured before, and after, aerosolization to determine whether aerosolization disrupted the activity of rAAT. It was determined that aerosolization did not significantly disrupt the integrity or activity of rAAT.
Vogelmeier et al., supra used the same techniques as Hubbard et al. in order to collect aerosolized secretory leukoprotease inhibitor (SLPI), i.e. bubbling through buffer in an impinger. To assess whether aerosolization had disrupted the activity of the SLPI, the time-concentration kinetics of neutrophil elastase inhibition were measured for aerosolized and un-aerosolized SLPI. Similar techniques to Hubbard et al. were adopted in order to measure the K.sub.a of SLPI for neutrophil elastase. The integrity of rSLPI after aerosolization was also evaluated using SDS-PAGE and Western analysis. Volgelmeier et al. found that aerosolization did not appear to alter the activity or integrity of SLPI.
While Hubbard et al. and Vogelmeier et al. discuss evaluation of the integrity and activity of the collected protein, they do not discuss the percentage of protein recovered. Collecting the aerosol by bubbling through buffer in an impinger may result in low collection efficiencies because it is likely that a significant fraction of the fine aerosol droplets will remain entrained in the airstream and not be collected. Thus, it is possible that the aerosolized protein collected by these methods, although fully active and intact, is not representative of the protein that is in the fine aerosol droplets escaping collection.
Colthorpe et al., Pharm. Res. 9:764-768 (1992), discuss collection of aerosolized insulin on a multistage liquid impinger operating at 60 L/min.sup.-1 with a reported collection efficiency of 92.5%. However, detailed analysis of protein integrity and activity was unavailable. A disadvantage associated with this method is the requirement for collection and analysis of multiple fractions to determine the effect nebulization has on protein integrity. Additionally, activity and integrity analysis of the collected protein on the terminal filter may not be possible due to surface adsorption or denaturation, drying, and difficulties encountered in re-solubilizing the entrapped protein.
It is, therefore, an object of the present invention to provide a method for collection of aerosolized proteins which facilitates enhanced recovery of the aerosolized protein when compared to earlier techniques and which enables the collected protein to be evaluated to assess the effect aerosolization has on the activity and integrity of the protein.
It is a further object of the present invention to provide a method of collecting nebulized therapeutic proteins, such as rhDNase, in order to assess the activity and integrity of the therapeutic protein which would be supplied by aerosol to the lungs of a patient.
Other objects and advantages of the present invention will become apparent to one of ordinary skill in the art.